Herpes simplex virus (HSV) is a human pathogen that is capable of both rapid productive infections, which lead to destruction of the infected host cells, and latent infections where the viral genome is relatively silent, persisting in the neurons of sensory ganglia for many years. HSV specifies a number of transacting proteins that alter the regulatory pathways and transcriptional machinery of the cell. The ICP4 protein of HSV is required for viral growth and is involved in the regulated transcription of nearly every viral gene. Like many transcriptional regulatory proteins, the ICP4 protein of herpes simplex virus (HSV) is modular in composition, consisting of multiple discrete domains that collectively contribute to its function during viral infection. Given our knowledge f the domain composition of ICP4, one aim of this proposal is to generate and study mutants of the ICP4 protein that specify a subset of functional domains. When individual functional domains are expressed, they often retain the ability to engage in the interactions that the intact protein normally requires for function, and therefore viral growth. Such mutant proteins are often transdominant inhibitors of viral growth. Transdominant mutant proteins may act by competing with the intact protein for productive sites of action, or by interacting with the intact protein through the normal process of dimerization, resulting in less active, or inactive, heterodimers. In addition, the overexpression of transactivation domains may lead to nonproductive interactions with the target proteins of ICP4, which are presumably cellular transcription factors. The proposed studies distinguish between these possibilities for various transdominant mutants by examining inhibition in the presence and absence of ICP4 and other viral proteins. Given the mechanism of action of specific transdominant proteins, experiments with the isolated proteins will be conducted to investigate and identify the cellular proteins that interact with ICP4 and are involved in ICP4 function. Preliminary data are presented implicating two such proteins: the human transcription general factor, TFIID, and protein kinase A. Much of this proposal is dedicated to tracking the regulatory circuitry involving HSV infection, the activity of protein kinase A, the involvement of ICP4 (and possibly other viral proteins) and the consequences for the activity of the general transcription machinery, specifically, TFIID. Transdominant mutants of both protein kinase A and human TFIID will be employed to help elucidate the interactions between ICP4, the relevant signal transduction pathways and the general transcriptional machinery functioning in infected cells in lytic and latent infection. We will explore inhibitory effects in vivo by constructing transgenic mice that express transdominant mutants in response to infection. The experiments with transgenic mice will also help determine the stage/s in the pathogenesis of HSV most susceptible to intervention through disruption of the interactions discussed above.